Stabilised Protein Solutions

ABSTRACT

The invention relates to a method of stabilizing highly concentrated protein solutions during ultrafiltration by addition of sucrose to the highly concentrated protein solution, in particular, but not exclusively to a method of stabilizing highly concentrated antibody solutions during ultrafiltration by addition of sucrose to the highly concentrated antibody solution.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.14/396,389, filed Oct. 23, 2014, which is a 35 U.S.C. § 371 NationalStage application of International Application PCT/EP2013/055390(WO2013/170977), filed Mar. 15, 2013, which claimed priority of EuropeanPatent Application 12167958.3, filed May 14, 2012; this applicationclaims priority under 35 U.S.C. § 119 of U.S. Provisional Application61/651,598; filed May 25, 2012; all of which are incorporated byreference.

DESCRIPTION

The current invention relates to ultrafiltration of concentrated proteincontaining solutions, particularly ultrafiltration of concentratedantibody containing solutions, particularly stabilisation of suchsolutions during ultrafiltration.

BACKGROUND

A number of injectable protein containing solutions, particular antibodycontaining solutions are supplied to the market. The use of thosesolutions for subcutaneous injection, results in demand of highconcentration of solutions due to limitations on injection volume.

General methods for purification of antibodies are well-known in the artand are for instance described in Pete Gagnon: Purification Tools formonoclonal Antibodies (1996) ISBN-9653515-9-9.

After purification of the antibody in solution, the concentration of theantibody is increased by use of ultrafiltration (UF) and then, at somepoint (usually around a concentration of 50 g/L antibody (mAb), theformulation buffer is diafiltrated into the solution by ultrafiltration,then the formulated solution is usually further concentrated to itsfinal concentration of between 100 g/L and 300 g/L, without sucrose,which normally will be added to the product after final ultrafiltrationconcentration. During concentration of protein/antibodies byultrafiltration, the protein concentration at the membrane surface, theso called wall concentration may increase to high levels. This can proveharmful to the protein/antibody and causes it to denature andprecipitate. This is often assumed to be the reason for observeddifferences in the calculated protein concentration and the measuredprotein concentration.

The problem with different concentration by calculation on basis ofreduction factors against the actual measured concentration by UV280 nmabsorbance in the retentate affects the yield of the ultrafiltrationprocess. The know solution to this problem is to try to recover theprotein by washing the module several times with buffers but therecovered protein will appear in greatly reduced concentrations.

It is therefore a need for a method that solves the problem ofdenaturation and precipitation of high concentrated protein solutionsduring ultrafiltration.

It has been shown that high concentration of sucrose in proteinsolutions can stabilize the protein in solution, especially againstaggregation during prolonged storage of such solutions. However, therisk of infections may have resulted in avoiding sucrose as a componentduring ultrafiltration or other purification or concentration steps.

SUMMARY

The present invention provides a method of ultrafiltration of a highlyconcentrated protein solution. The present invention furthermoreprovides a method of stabilizing a highly concentrated protein solutionduring ultrafiltration.

The present invention provides a method of ultrafiltration of a highlyconcentred protein solution herein the protein solution is stabilizedduring the ultrafiltration by addition of sucrose to the solution. Thepresent invention furthermore provides a method of ultrafiltration of ahighly concentred protein solution, wherein the protein is an antibody.

DESCRIPTION

The present invention provides a method of ultrafiltration of a highlyconcentrated protein solution. The present invention furthermoreprovides a method of stabilizing a highly concentrated protein solutionduring ultrafiltration.

It has surprisingly been discovered that the addition of sucrose to ahighly concentrated protein solution before further concentration byultrafiltration seems to protect the protein from denaturating andprecipitating during ultrafiltration. This results in higher yield orrecovery % of the protein and less turbidity and formation ofaggregates, as measured by % HMWP during the ultrafiltration.

The term “protein”, “polypeptide” and “peptide” as used herein means acompound composed of at least five constituent amino acids connected bypeptide bonds. The constituent amino acids may be from the group of theamino acids encoded by the genetic code and they may be natural aminoacids which are not encoded by the genetic code, as well as syntheticamino acids. Natural amino acids which are not encoded by the geneticcode are e.g. hydroxyproline, y-carboxyglutamate, ornithine,phosphoserine, D-alanine and D-glutamine. Synthetic amino acids compriseamino acids manufactured by chemical synthesis, i.e. D-isomers of theamino acids encoded by the genetic code such as D-alanine and D-leucine,Aib (a-aminoisobutyric acid), Abu (α-aminobutyric acid), Tle(tert-butylglycine), β-alanine, 3-aminomethyl benzoic acid andanthranilic acid.

The term “antibody” and/or “mAb” as used herein covers monoclonalantibodies (including full length antibodies which have animmunoglobulin Fc region), antibody compositions with polyepitopicspecificity, bispecific antibodies, diabodies, and single-chainmolecules, as well as antibody fragments (e.g., Fab, F(ab′)₂, and Fv).

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. In addition to their specificity, the monoclonal antibodies areadvantageous in that they are synthesized by the hybridoma culture,uncontaminated by other immunoglobulins. The modifier “monoclonal”indicates the character of the antibody as being obtained from asubstantially homogeneous population of antibodies, and is not to beconstrued as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by the hybridoma method firstdescribed by Kohler et al., Nature, 256: 495 (1975), or may be made byrecombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The“monoclonal antibodies” may also be isolated from phage antibodylibraries using the techniques described in Clackson et al., Nature,352: 624-628 (1991) and Marks et al., J. Mol. Biol., 222: 581-597(1991), for example.

The monoclonal antibodies herein may extend to include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of the chain(s) is (are) identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567;Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)).

Examples of suitable antibodies, which may be formulated in a stablecomposition of the invention include: 3F8, Abagovomab, Abciximab, ACZ885(canakinumab), Adalimumab, Adecatumumab, Afelimomab, Afutuzumab,Alacizumab pegol, Alemtuzumab, Altumomab pentetate, Anatumomabmafenatox, Anrukinzumab (IMA-638), Apolizumab, Arcitumomab, Aselizumab,Atlizumab (tocilizumab), Atorolimumab, Bapineuzumab, Basiliximab,Bavituximab, Bectumomab, Belimumab, Bertilimumab, Besilesomab,Bevacizumab, Biciromab, Bivatuzumab mertansine, Blinatumomab,Brentuximab vedotin, Briakinumab, Canakinumab, Cantuzumab mertansine,Capromab pendetide, Catumaxomab, Cedelizumab, Certolizumab pegol,Cetuximab, Citatuzumab bogatox, Cixutumumab, Clenoliximab, Clivatuzumabtetraxetan, ONTO 148 (golimumab), ONTO 1275 (ustekinumab), Conatumumab,Dacetuzumab, Daclizumab, Denosumab, Detumomab, Dorlimomab aritox,Dorlixizumab, Ecromeximab, Eculizumab, Edobacomab, Edrecolomab,Efalizumab, Efungumab, Elsilimomab, Enlimomab pegol, Epitumomabcituxetan, Epratuzumab, Erlizumab, Ertumaxomab, Etaracizumab,Exbivirumab, Fanolesomab, Faralimomab, Felvizumab, Fezakinumab,Figitumumab, Fontolizumab, Foravirumab, Fresolimumab, Galiximab,Gantenerumab, Gavilimomab, Gemtuzumab ozogamicin, Golimumab,Gomiliximab, Ibalizumab, Ibritumomab tiuxetan, Igovomab, lmciromab,Infliximab, Intetumumab, Inolimomab, Inotuzumab ozogamicin, Ipilimumab,Iratumumab, Keliximab, Labetuzumab, Lebrikizumab, Lemalesomab,Lerdelimumab, Lexatumumab, Libivirumab, Lintuzumab, Lucatumumab,Lumiliximab, Mapatumumab, Maslimomab, Matuzumab, Mepolizumab,Metelimumab, Milatuzumab, Minretumomab, Mitumomab, Morolimumab,Motavizumab, Muromonab-CD3, MYO-029 (stamulumab), Nacolomab tafenatox,Naptumomab estafenatox, Natalizumab, Nebacumab, Necitumumab,Nerelimomab, Nimotuzumab, Nofetumomab merpentan, Ocrelizumab,Odulimomab, Ofatumumab, Omalizumab, Oportuzumab monatox, Oregovomab,Otelixizumab, Pagibaximab, Palivizumab, Panitumumab, Panobacumab,Pascolizumab, Pemtumomab, Pertuzumab, Pexelizumab, Pintumomab,Priliximab, Pritumumab, PRO 140, Rafivirumab, Ramucirumab, Ranibizumab,Raxibacumab, Regavirumab, Reslizumab, Rilotumumab, Rituximab,Robatumumab, Rontalizumab, Rovelizumab, Ruplizumab, Satumomab,Sevirumab, Sibrotuzumab, Sifalimumab, Siltuximab, Siplizumab,Solanezumab, Sonepcizumab, Sontuzumab, Stamulumab, Sulesomab,Tacatuzumab tetraxetan, Tadocizumab, Talizumab, Tanezumab, Taplitumomabpaptox, Tefibazumab, Telimomab aritox, Tenatumomab, Teneliximab,Teplizumab, TGN1412, Ticilimumab (tremelimumab), Tigatuzumab, TNX-355(ibalizumab), TNX-650, TNX-901 (talizumab), Tocilizumab (atlizumab),Toralizumab, Tositumomab, Trastuzumab, Tremelimumab, Tucotuzumabcelmoleukin, Tuvirumab, Urtoxazumab, Ustekinumab, Vapaliximab,Vedolizumab, Veltuzumab, Vepalimomab, Visilizumab, Volociximab,Votumumab, Zalutumumab, Zanolimumab, Ziralimumab, Zolimomab aritox andthe like.

In one embodiment, the protein is an immunoglobulin. In one embodiment,the protein is an antibody. In one embodiment, the protein is amonoclonal antibody (mAb). In one embodiment, the protein is an IgG4antibody.

In one embodiment, the antibody is a monoclonal anti-IL20 antibody. Inone embodiment, the antibody is an anti-IL20 antibody as described inWO2010/000721. In one embodiment, the anti-IL20 monoclonal antibody is15D2 or 5B7 as described in WO2010/000721.

It will be appreciated that the invention finds particular utility wherethe protein is present in solution to be ultrafiltrated in highconcentrations. Thus, in one embodiment, the protein is present in aconcentration of 40 g/L or more in the solution to be ultrafiltrated,such as 40, 45, 40, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120,130, 140, 150, 175, 200, 250, 300 g/L or more.

The final ultrafiltration retentate will have higher proteinconcentrations, such as in an amount of between 75 g/L and 400 g/L, forinstance between 75 g/L and 350 g/L, such as between 75 g/L and 300 g/L,for instance between 100 g/L and 250 g/L, such as between 75 g/L and 200g/L, for instance between 75 g/L and 150 g/L, such as between 75 g/L and100 g/L. In one embodiment, the protein is present in theultrafiltration retentate in a concentration of between 100 g/L and 400g/L, for instance between 100 g/L and 350 g/L, such as between 100 g/Land 300 g/L, for instance between 100 g/L and 250 g/L, such as between100 g/L and 200 g/L, for instance between 100 g/L and 150 g/L. In oneembodiment, the protein is present in the ultrafiltration retentate aconcentration of between 125 g/L and 400 g/L, for instance between 125g/L and 350 g/L, such as between 125 g/L and 300 g/L, for instancebetween 125 g/L and 250 g/L, such as between 125 g/L and 200 g/L, forinstance between 125 g/L and 150 g/L. In one embodiment, the protein ispresent in a concentration between 150 g/L and 400 g/L, such as between150 g/L and 350 g/L, for instance between 150 g/L and 300 g/L, such asbetween 150 g/L and 250 g/L, for instance between 150 g/L and 200 g/L.

The term “stability” of a protein in a composition as used herein refersto the biological stability, physical stability or chemical stability ofthe protein in solution. Chemical covalent changes in the proteinstructure leading to formation of chemical degradation products withpotential less biological potency and/or potential increased immunogenicproperties compared to the native protein structure, duringmanufacturing process. Various chemical degradation products can beformed depending on the type and nature of the native protein and theenvironment to which the protein is exposed. Elimination of chemicaldegradation can most probably not be completely avoided and increasingamounts of chemical degradation products is often seen during storageand use of the protein composition is well-known by the person skilledin the art. Most proteins are prone to deamidation, a process in whichthe side chain amide group in glutaminyl or asparaginyl residues ishydrolysed to form a free carboxylic acid. Other degradation pathwaysinvolve formation of high molecular weight transformation products wheretwo or more protein molecules are covalently bound to each other throughtransamidation and/or disulfide interactions leading to formation ofcovalently bound dimer, oligomer and polymer degradation products(Stability of Protein Pharmaceuticals, Ahern. T. J. & Manning M. C.,Plenum Press, New York 1992). Oxidation (of for instance methionineresidues) can be mentioned as another variant of chemical degradation.The chemical stability of the protein composition can be evaluated bymeasuring the amount of the chemical degradation products at varioustime-points after exposure to different environmental conditions (theformation of degradation products can often be accelerated by forinstance increasing temperature). The amount of each individualdegradation product is often determined by separation of the degradationproducts depending on molecule size and/or charge using variouschromatography techniques (e.g. SEC-HPLC and/or RP-HPLC).

There are various analytical techniques for measuring protein stabilityavailable in the art (Peptide and Protein Drug Delivery, 247-301,Vincent Lee Ed. & Marcel Dekker, N.Y. Pubs 1991; and Jones, A. Adv. DrugDelivery Rev. 10: 29-90, 1993).

SEC-H PLC is in particular used for quantification of proteinaggregates. The samples may for instance be analysed using a TSK G3000SWXL column, isocratic elution and subsequent UV detection at 214 or 280nm. This method is used to determine monomeric IgG content and % HighMolecular Weight Proteins (HMWP) consisting of dimeric species or largerwhich are separated according to size by the gel resin. The monomericcontent and % HMWP are determined relative to the total protein contentdetected by the method.

Physical stability of protein solution can be measured by well-knownmethods, including measurement of attenuation of light by measurement ofabsorbance or optical density. Such measurements relate to the turbidityof solution.

The term “turbidity” of a solution as used herein refer to the presenceof cloudiness or haze in the protein solution. In protein solutions,turbidity is typically measured using a UV-visible spectrophotometer atwavelengths between 320-800 nm. The degree of turbidity can becalculated by reference to a standard curve generated by solutions withknown turbidity. For protein containing pharmaceutical solutions,reference standards can be based on the European Pharmacopeia (Ph. Eur.)Section 2.2.1, that defines visual clarity and describes standard levelsof turbidity in relation to water.

The term ultrafiltration (UF) as used herein refers to a variety ofmembrane filtration in which hydrostatic pressure forces a liquid or asolution against a semipermeable membrane. Solutes of high molecularweight are retained (retentate), while water and low molecular weightsolutes pass through the membrane (permeate or filtrate). Thisseparation process is used in industry and research for purifying andconcentrating macromolecular (10³-10⁶ Da) solutions, especially proteinsolutions. Ultrafiltration is especially applied in cross-flowfiltration mode. The term retentate, as used herein, refers to what isretained, that is the portion or content of the liquid or solution thatdoes not pass through the ultrafiltration membrane. The term filtrate,as used herein, refers to the portion or part of the liquid or solutionthat does pass the membrane during ultrafiltration and is not retained.

Concentration factor is a number which is reciprocal to the volumereduction of the retentate during ultrafiltration concentration. Whenthe retentate volume is reduced to ½, the concentration factor will be2. The term “calculated concentration” as used herein refers to anestimated concentration that is calculated by multiplying the originalmAb concentration by the concentration factor.

The term “yield” or “recovery %” as used herein, refers to the amount ofprotein recovered in during the ultrafiltration step or steps, in theretentate or retentates if multiply ultrafiltration or washing stepshave been performed, compared with the total amount of protein in thestarting protein solution to be ultrafiltrated.

The term cross-flow filtration as used herein refers to a proteinpurification method, also known as tangential flow filtration that is atype of filtration (a particular unit operation). Cross-flow filtrationis different from dead-end filtration in which the feed is passedthrough a membrane or bed, the solids being trapped in the filter andthe filtrate being released at the other end. Cross-flow filtration getsits name because the majority of the feed flow travels tangentiallyacross the surface of the filter, rather than into the filter. Theprincipal advantage of this is that the filter cake (which can blind thefilter) is substantially washed away during the filtration process,increasing the length of time that a filter unit can be operational. Itcan be a continuous process, unlike batch-wise dead-end filtration.

In cross-flow filtration, the feed is passed across the filter membrane(tangentially) at positive pressure relative to the permeate side. Aproportion of the material which is smaller than the membrane pore sizepasses through the membrane as permeate or filtrate; everything else isretained on the feed side of the membrane as retentate.

The volume of the fluid is reduced by allowing permeate flow to occur.Solvent, solutes, and particles smaller than the membrane pore size passthrough the membrane, while particles larger than the pore size areretained, and thereby concentrated. In bioprocessing applications,concentration may be followed by diafiltration.

The term diafiltration, as used herein, refers to a dilution andre-concentration of the solution or liquid, where in order toeffectively remove permeate components from the solution or liquid,fresh solvent may be added to the feed to replace the permeate volume,at the same rate as the permeate flow rate, such that the volume in thesystem remains constant. This is analogous to the washing of filter caketo remove soluble components.

EXAMPLES Example 1

In order to perform an optimisation programme on the ULTRAFILTRATION ofan solution containing Anti-IL-20 antibody, solution A was diafiltratedagainst solution B (5× retentate volume) on a Äkta crossflow equippedwith a Pellicon 3, Ultracel 30 K membrane, 88 cm2 module (Millipore).

Solution A:

100 g/L Anti IL20

150 mM Sucrose

25 mM Arg

25 mM NaCl

33 mM His

pH 6.5

Solution B:

25 mM Arg

25 mM NaCl

33 mM His

pH 6.5

The Äkta crossflow was also equipped with a heat exchanger (Exergy,series 17 model00402) used to regulate the retentate temperature to 45C. Delta P (Pin-Pout) was 2 bar and TMP (Trans membrane pressure) was 1bar. After diafiltration the retentate was concentrated withultrafiltration according to Table 1.

The effect of diafiltration was that the appearance of the solutionturned from clear to milky, indicating precipitation of the protein.

Concentration factor is a number which is reciprocal to the volumereduction of the retentate during ultrafiltration concentration. Whenthe retentate volume is reduced to ½, the concentration factor will be2. This factor is used to determine the calculated mAb concentration bymultiplying with the original concentration: 108.3×2=216.6 During thefollowing concentration of anti-IL20 solutions in solution B with andwithout 150 mM sucrose the mAb content was measured and compared to thecalculated content (calculated by concentration factor), Table 1.

TABLE 1 Comparison of calculated concentration versus measured (actual)concentration, with and without addition of sucrose. Concentration withConcentration without sucrose (Solution A) sucrose (Solution B) Cal-Concen- Cal- Concen- culated Measured tration culated Measured trationconc.(g/L) conc.(g/L) factor conc.(g/L) conc.(g/L) factor 108.3 108.5* 191.8 77.8* 1 216.6 215.8 2 209.3 136.7 2.28 270.8 243.1 2.5 229.5 145.32.5 359.55 303.3 3.32 275.4 162.6 3 325.0 183.7 3.54 372.7 197.9 4.06(*The fact that there is difference between measure and calculatedconcentrations already with a concentration factor 1 is a result ofprecipitation during diafiltration.)

This shows a difference in the calculated versus measured concentrationswhich could be explained by creation of a build-up layer of extremelyconcentrated protein at the membrane surface, maybe fouled orprecipitated protein or protein aggregates. The difference is muchlarger when sucrose is not present.

Example 2

In order to investigate the effect addition of sucrose beforeultrafiltration, on the formation of HMWP (High Molecular WeightProteins: aggregates) during ultrafiltration the retentate contentmentof HMWP was measured including some samples from washing of the cassetteafter emptying for product as can be seen the washing fractions are ofsubstantially lower concentrations than the original retentate. The HMWPcontent of the wash fraction is interesting because it shows theconstitution of the gel—or build up layer material which is not shown inthe retentate. The overall result is that the material with sucrosepresent during concentration has lower content of HMWP in accordancewith the theory that sucrose protects the mAb.

Content of mAb was determined by UV280 nm absorption with a NanoDrop2000C instrument (Thermo Scientific) and the HMWP was determined byanalytical a SEC (Size Exclusion Chromatography) HPLC method with a TSKGel G3000SW_(XL) 7.8×300 mm column.

TABLE 2 The HMPW content and mAb concentrations of retentate and washingportions from ultrafiltration of solution B (without sucrose).Concentration Sample ID (g/L) HMWP (%) Retentate 174 1.2 Wash 1 108 1.2Wash 2 67 1.1 Wash 3 41 1.1

TABLE 3 HMPW content and mAb concentrations of retentate and washingportions from ultrafiltration of solution A (with sucrose).Concentration Sample ID (g/L) HMWP (%) Retentate 296 1.0 Wash 1 148 1.0Wash 2 95 1.0 Wash 3 59 0.9 Wash 4 26 0.9

It may be concluded from both Table 1. and Tables 2. and 3. that theimproved correlation between measured and calculated retentateconcentrations during ultrafiltration concentration of solution A andthe results from HMWP analysis indicate that the presence of sucroseprevents to some extent the aggregation of the protein and the creationof build-up layer (or fouling).

Example 3

In order to investigate the effect addition of sucrose beforeultrafiltration, on the protein recovery or yield of the process, theconcentration of mAb was measured in the retentate and samples fromwashing of the cassette after emptying for product.

Content of mAb was determined by UV280 nm absorption with a NanoDrop2000C instrument (Thermo Scientific).

Table 4. shows the yield or recovery of the anti-IL-20 antibody fromsolution B retentate and samples of the cassette. The starting volumewas 140 ml, with an antibody concentration of 107.45 g/L, or in total 15g of antibody.

TABLE 4 Yield of antibody in retentate and washing portions fromultrafiltration of solution B (without sucrose). Volume ConcentrationmAb Sample ID (ml) (g/L) (g) Retentate 37.5 192.2 7.2 Wash 1 22 118.92.6 Wash 2 24 82.1 2.0 Wash 3 50 48.3 2.2 Total 14.0 Total yield 93.0%

Table 5. shows the yield or recovery of anti-IL-20 from solution A. Thestarting volume was 300 ml, with an antibody concentration of 107.45 g/Lor 32.2 g of antibody in total.

TABLE 5 Yield of antibody in retentate and washing portions fromultrafiltration of solution A (with sucrose). Volume Concentration mAbSample ID (ml) (g/L) (g) Retentate 68 303.28 20.6 Wash 1 24 90 2.2 Wash2 23 171.1 3.9 Wash 3 23 105.5 2.4 Wash 4 23 66 1.5 Wash 5 50 29.8 1.5Total 32.2 Total yield 99.8%

The following is a non-limiting list of embodiments of the presentinvention.

1. A method of ultrafiltration of a highly concentrated proteinsolution, wherein sucrose is added to the solution when the proteinconcentration of the solution is between 40 and 60 g/L before furtherconcentration by ultrafiltration.

2. A method of decreasing the difference between a calculated orestimated protein concentration and actual measured proteinconcentration in a highly concentrated protein ultrafiltrationretentate, wherein sucrose is added to the solution when the proteinconcentration of the solution is between 40 g/L and 60 g/L beforefurther concentration by ultrafiltration.

3. A method of decreasing the difference between a calculated orestimated protein concentration and an actual measured proteinconcentration in a highly concentrated protein ultrafiltration retentateto less than 15%, wherein sucrose is added to the solution when theprotein concentration of the solution is between 40 and 60 g/L beforefurther concentration by ultrafiltration.

4. A method of decreasing the difference between a calculated orestimated protein concentration and an actual measured proteinconcentration in a highly concentrated protein ultrafiltration retentateto less than 20%, wherein sucrose is added to the solution when theprotein concentration of the solution is between 40 and 60 g/L beforefurther concentration by ultrafiltration.

5. A method of decreasing the difference between a calculated protein orestimated concentration and an actual measured protein concentration ina highly concentrated protein ultrafiltration retentate to less than25%, wherein sucrose is added to the solution when the proteinconcentration of the solution is between 40 and 60 g/L before furtherconcentration by ultrafiltration.

6. A method of decreasing the difference between a calculated orestimated protein concentration and an actual measured proteinconcentration in a highly concentrated protein ultrafiltration to lessthan 30%, wherein sucrose is added to the solution when the proteinconcentration of the solution is between 40 and 60 g/L before furtherconcentration by ultrafiltration.

7. A method of ultrafiltration of a highly concentrated proteinsolution, where the difference between the calculated or estimatedprotein concentration and the actual measured protein concentration inthe highly concentrated ultrafiltration retentate is less than 15%,wherein sucrose is added to the solution when the protein concentrationof the solution is between 40 and 60 g/L before further concentration byultrafiltration.

8. A method of ultrafiltration of a highly concentrated proteinsolution, where the difference between the calculated or estimatedprotein concentration and the actual measured protein concentration inthe highly concentrated ultrafiltration retentate is less than 20%,wherein sucrose is added to the solution when the protein concentrationof the solution is between 40 and 60 g/L before further concentration byultrafiltration.

9. A method of ultrafiltration of a highly concentrated proteinsolution, where the difference between the calculated or estimatedprotein concentration and the actual measured protein concentration inthe highly concentrated ultrafiltration retentate is less than 25%,wherein sucrose is added to the solution when the protein concentrationof the solution is between 40 and 60 g/L before further concentration byultrafiltration.

10. A method of ultrafiltration of a highly concentrated proteinsolution, where the difference between the calculated or estimatedprotein concentration and the actual measured protein concentration inthe highly concentrated ultrafiltration retentate is less than 30%,wherein sucrose is added to the solution when the protein concentrationof the solution is between 40 and 60 g/L before further concentration byultrafiltration.

11. A method of increasing the recovery of protein by ultrafiltration ofa highly concentrated protein solution, wherein sucrose is added to thesolution when the protein concentration of the solution is between 40and 60 g/L before further concentration by ultrafiltration.

12. A method of increasing the recovery of protein by ultrafiltration ofa highly concentrated protein solution to above 94% of the total amountof said protein in the solution to be ultrafiltrated, wherein sucrose isadded to the solution when the protein concentration of the solution isbetween 40 and 60 g/L before further concentration by ultrafiltration.

13. A method of increasing the recovery of protein by ultrafiltration ofa highly concentrated protein solution to above 95% of the total amountof said protein in the solution to be ultrafiltrated, wherein sucrose isadded to the solution when the protein concentration of the solution isbetween 40 and 60 g/L before further concentration by ultrafiltration.

14. A method of increasing the recovery of protein by ultrafiltration ofa highly concentrated protein solution to above 96% of the total amountof said protein in the solution to be ultrafiltrated, wherein sucrose isadded to the solution when the protein concentration of the solution isbetween 40 and 60 g/L before further concentration by ultrafiltration.

15. A method of increasing the recovery of protein by ultrafiltration ofa highly concentrated protein solution to above 97% of the total amountof said protein in the solution to be ultrafiltrated, wherein sucrose isadded to the solution when the protein concentration of the solution isbetween 40 and 60 g/L before further concentration by ultrafiltration.

16. A method of increasing the recovery of protein by ultrafiltration ofa highly concentrated protein solution to above 98% of the total amountof said protein in the solution to be ultrafiltrated, wherein sucrose isadded to the solution when the protein concentration of the solution isbetween 40 and 60 g/L before further concentration by ultrafiltration.

17. A method of increasing the recovery of protein by ultrafiltration ofa highly concentrated protein solution to above 99% of the total amountof said protein in the solution to be ultrafiltrated, wherein sucrose isadded to the solution when the protein concentration of the solution isbetween 40 and 60 g/L before further concentration by ultrafiltration.

18. A method of ultrafiltration of a highly concentrated proteinsolution, where the recovery of protein by ultrafiltration is above 95%of the total amount of said protein in the solution to beultrafiltrated, wherein sucrose is added to the solution when theprotein concentration of the solution is between 40 and 60 g/L beforefurther concentration by ultrafiltration.

19. A method of ultrafiltration of a highly concentrated proteinsolution, where the recovery of protein by ultrafiltration is above 96%of the total amount of said protein in the solution to beultrafiltrated, wherein sucrose is added to the solution when theprotein concentration of the solution is between 40 and 60 g/L beforefurther concentration by ultrafiltration.

20. A method of ultrafiltration of a highly concentrated proteinsolution, where the recovery of protein by ultrafiltration is above 97%of the total amount of said protein in the solution to beultrafiltrated, wherein sucrose is added to the solution when theprotein concentration of the solution is between 40 and 60 g/L beforefurther concentration by ultrafiltration.

21. A method of ultrafiltration of a highly concentrated proteinsolution, where the recovery of protein by ultrafiltration is above 98%of the total amount of said protein in the solution to beultrafiltrated, wherein sucrose is added to the solution when theprotein concentration of the solution is between 40 and 60 g/L beforefurther concentration by ultrafiltration.

22. A method of ultrafiltration of a highly concentrated proteinsolution, where the recovery of protein by ultrafiltration is above 99%of the total amount of said protein in the solution to beultrafiltrated, wherein sucrose is added to the solution when theprotein concentration of the solution is between 40 and 60 g/L beforefurther concentration by ultrafiltration.

23. A method of increasing the recovery of protein by ultrafiltration ofa highly concentrated protein solution in the final ultrafiltrationretentate to above 50% of the total amount of said protein in thesolution to be ultrafiltrated, wherein sucrose is added to the solutionwhen the protein concentration of the solution is between 40 and 60 g/Lbefore further concentration by ultrafiltration.

24. A method of increasing the recovery of protein by ultrafiltration ofa highly concentrated protein solution in the final ultrafiltrationretentate to above 55% of the total amount of said protein in thesolution to be ultrafiltrated, wherein sucrose is added to the solutionwhen the protein concentration of the solution is between 40 and 60 g/Lbefore further concentration by ultrafiltration.

25. A method of increasing the recovery of protein by ultrafiltration ofa highly concentrated protein solution in the final ultrafiltrationretentate to above 60% of the total amount of said protein in thesolution to be ultrafiltrated, wherein sucrose is added to the solutionwhen the protein concentration of the solution is between 40 and 60 g/Lbefore further concentration by ultrafiltration.

26. A method of ultrafiltration of a highly concentrated proteinsolution, where the recovery of protein by ultrafiltration in the finalultrafiltration retentate is above 50% of the total amount of saidprotein in the solution to be ultrafiltrated, wherein sucrose is addedto the solution when the protein concentration of the solution isbetween 40 and 60 g/L before further concentration by ultrafiltration.

27. A method of ultrafiltration of a highly concentrated proteinsolution, where the recovery of protein by ultrafiltration in the finalultrafiltration retentate is above 55% of the total amount of saidprotein in the solution to be ultrafiltrated, wherein sucrose is addedto the solution when the protein concentration of the solution isbetween 40 and 60 g/L before further concentration by ultrafiltration.

28. A method of ultrafiltration of a highly concentrated proteinsolution, where the recovery of protein by ultrafiltration in the finalultrafiltration retentate is above 60% of the total amount of saidprotein in the solution to be ultrafiltrated, wherein sucrose is addedto the solution when the protein concentration of the solution isbetween 40 and 60 g/L before further concentration by ultrafiltration.

29. A method of stabilizing a highly concentrated protein solutionduring ultrafiltration, wherein sucrose is added to the solution whenthe protein concentration of the solution is between 40 and 60 g/Lbefore further concentration by ultrafiltration.

30. A method of stabilizing a highly concentrated protein solutionduring ultrafiltration, where the level of HMWP aggregates is 1% orbelow 1%, wherein sucrose is added to the solution when the proteinconcentration of the solution is between 40 and 60 g/L before furtherconcentration by ultrafiltration.

31. A method of stabilizing a highly concentrated protein solutionduring ultrafiltration, where the level of HMWP aggregates is below1.1%, wherein sucrose is added to the solution when the proteinconcentration of the solution is between 40 and 60 g/L before furtherconcentration by ultrafiltration.

32. A method of decreasing the formation of HWMP aggregates in a highlyconcentrated protein solution during ultrafiltration, wherein sucrose isadded to the solution when the protein concentration of the solution isbetween 40 and 60 g/L before further concentration by ultrafiltration.

33. A method of decreasing the formation of HWMP aggregates in a highlyconcentrated protein solution during ultrafiltration to 1% or below 1%,wherein sucrose is added to the solution when the protein concentrationof the solution is between 40 and 60 g/L before further concentration byultrafiltration.

34. A method of decreasing the formation of HWMP aggregates in a highlyconcentrated protein solution during ultrafiltration to below 1.1%,wherein sucrose is added to the solution when the protein concentrationof the solution is between 40 and 60 g/L before further concentration byultrafiltration.

35. A method of ultrafiltration of a highly concentrated proteinsolution, where the level of HMWP aggregates is kept at 1% or below 1%,wherein sucrose is added to the solution when the protein concentrationof the solution is between 40 and 60 g/L before further concentration byultrafiltration.

36. A method of ultrafiltration of a highly concentrated proteinsolution, where the level of HMWP aggregates is kept below 1.1%, whereinsucrose is added to the solution when the protein concentration of thesolution is between 40 and 60 g/L before further concentration byultrafiltration.

37. A method of stabilizing a highly concentrated protein solutionduring ultrafiltration, where the formation of HMWP aggregates duringultrafiltration is decreased by 10%, wherein sucrose is added to thesolution when the protein concentration of the solution is between 40and 60 g/L before further concentration by ultrafiltration.

38. A method of stabilizing a highly concentrated protein solutionduring ultrafiltration, where the formation of HMWP aggregates duringultrafiltration is decreased by 20%, wherein sucrose is added to thesolution when the protein concentration of the solution is between 40and 60 g/L before further concentration by ultrafiltration.

39. A method of ultrafiltration of a highly concentrated proteinsolution, where the formation of HMWP aggregates is decreased by 10%,wherein sucrose is added to the solution when the protein concentrationof the solution is between 40 and 60 g/L before further concentration byultrafiltration.

40. A method of ultrafiltration of a highly concentrated proteinsolution, where the formation of HMWP aggregates is decreased by 20%,wherein sucrose is added to the solution when the protein concentrationof the solution is between 40 and 60 g/L before further concentration byultrafiltration.

41. A method according to any of embodiments 1-40, wherein the proteinconcentration is 40 g/L when sucrose is added to the solution.

42. A method according to any of embodiments 1-40, wherein the proteinconcentration is 41 g/L when sucrose is added to the solution.

43. A method according to any of embodiments 1-40, wherein the proteinconcentration is 42 g/L when sucrose is added to the solution.

44. A method according to any of embodiments 1-40, wherein the proteinconcentration is 43 g/L when sucrose is added to the solution.

45. A method according to any of embodiments 1-40, wherein the proteinconcentration is 44 g/L when sucrose is added to the solution.

46. A method according to any of embodiments 1-40, wherein the proteinconcentration is 45 g/L when sucrose is added to the solution.

47. A method according to any of embodiments 1-40, wherein the proteinconcentration is 46 g/L when sucrose is added to the solution.

48. A method according to any of embodiments 1-40, wherein the proteinconcentration is 47 g/L when sucrose is added to the solution.

49. A method according to any of embodiments 1-40, wherein the proteinconcentration is 48 g/L when sucrose is added to the solution.

50. A method according to any of embodiments 1-40, wherein the proteinconcentration is 49 g/L when sucrose is added to the solution.

51. A method according to any of embodiments 1-40, wherein the proteinconcentration is 50 g/L when sucrose is added to the solution.

52. A method according to any of embodiments 1-40, wherein the proteinconcentration is 51 g/L when sucrose is added to the solution.

53. A method according to any of embodiments 1-40, wherein the proteinconcentration is 52 g/L when sucrose is added to the solution.

54. A method according to any of embodiments 1-40, wherein the proteinconcentration is 53 g/L when sucrose is added to the solution.

55. A method according to any of embodiments 1-40, wherein the proteinconcentration is 54 g/L when sucrose is added to the solution.

56. A method according to any of embodiments 1-40, wherein the proteinconcentration is 55 g/L when sucrose is added to the solution.

57. A method according to any of embodiments 1-40, wherein the proteinconcentration is 56 g/L when sucrose is added to the solution.

58. A method according to any of embodiments 1-40, wherein the proteinconcentration is 57 g/L when sucrose is added to the solution.

59. A method according to any of embodiments 1-40, wherein the proteinconcentration is 58 g/L when sucrose is added to the solution.

60. A method according to any of embodiments 1-40, wherein the proteinconcentration is 59 g/L when sucrose is added to the solution.

61. A method according to any of embodiments 1-40, wherein the proteinconcentration is 60 g/L when sucrose is added to the solution.

62. A method according to any of embodiments 1-40, wherein the proteinconcentration is between 40 g/L and 45 g/L when sucrose is added to thesolution.

63. A method according to any of embodiments 1-40, wherein the proteinconcentration is between 40 g/L and 50 g/L when sucrose is added to thesolution.

64. A method according to any of embodiments 1-40, wherein the proteinconcentration is between 45 g/L and 50 g/L when sucrose is added to thesolution.

65. A method according to any of embodiments 1-40, wherein the proteinconcentration is between 45 g/L and 55 g/L when sucrose is added to thesolution.

66. A method according to any of embodiments 1-40, wherein the proteinconcentration is between 50 g/L and 55 g/L when sucrose is added to thesolution.

67. A method according to any of embodiments 1-40, wherein the proteinconcentration is between 50 g/L and 60 g/L when sucrose is added to thesolution.

68. A method according to any of embodiments 1-40, wherein the proteinconcentration is between 55 g/L and 60 g/L when sucrose is added to thesolution.

69. A method according to any of embodiments 1-68, wherein the sucroseis added in the concentration of between 50 mM and 300 mM.

70. A method according to any of embodiments 1-68, wherein the sucroseis added in the concentration of between 50 mM and 250 mM.

71. A method according to any of embodiments 1-68, wherein the sucroseis added in the concentration of between 50 mM and 200 mM.

72. A method according to any of embodiments 1-68, wherein the sucroseis added in the concentration of between 50 mM and 150 mM.

73. A method according to any of embodiments 1-68, wherein the sucroseis added in the concentration of between 50 mM and 100 mM.

74. A method according to any of embodiments 1-68, wherein the sucroseis added in the concentration of between 100 mM and 300 mM.

75. A method according to any of embodiments 1-68, wherein the sucroseis added in the concentration of between 100 mM and 250 mM.

76. A method according to any of embodiments 1-68, wherein the sucroseis added in the concentration of between 100 mM and 200 mM.

77. A method according to any of embodiments 1-68, wherein the sucroseis added in the concentration of between 100 mM and 150 mM.

78. A method according to any of embodiments 1-68, wherein the sucroseis added in the concentration of 100 mM.

79. A method according to any of embodiments 1-68, wherein the sucroseis added in the concentration of 110 mM.

80. A method according to any of embodiments 1-68, wherein the sucroseis added in the concentration of 120 mM.

81. A method according to any of embodiments 1-68, wherein the sucroseis added in the concentration of 130 mM.

82. A method according to any of embodiments 1-68, wherein the sucroseis added in the concentration of 140 mM.

83. A method according to any of embodiments 1-68, wherein the sucroseis added in the concentration of 150 mM.

84. A method according to any of embodiments 1-68, wherein the sucroseis added in the concentration of 160 mM.

85. A method according to any of embodiments 1-68, wherein the sucroseis added in the concentration of 175 mM.

86. A method according to any of embodiments 1-68, wherein the sucroseis added in the concentration of 200 mM.

87. A method according to any of embodiments 1-68, wherein the sucroseis added in the concentration of 225 mM.

88. A method according to any of embodiments 1-68, wherein the sucroseis added in the concentration of 250 mM.

89. A method according to any of embodiments 1-68, wherein the sucroseis added in the concentration of 300 mM.

90. A method of ultrafiltration of a highly concentrated antibodysolution, wherein sucrose is added to the solution when the antibodyconcentration of the solution is between 40 and 60 g/L before furtherconcentration by ultrafiltration.

91. A method of decreasing the difference between a calculated orestimated antibody concentration and actual measured antibodyconcentration in a highly concentrated antibody ultrafiltrationretentate, wherein sucrose is added to the solution when the antibodyconcentration of the solution is between 40 g/L and 60 g/L beforefurther concentration by ultrafiltration.

92. A method of decreasing the difference between a calculated orestimated antibody concentration and an actual measured antibodyconcentration in a highly concentrated antibody ultrafiltrationretentate to less than 15%, wherein sucrose is added to the solutionwhen the antibody concentration of the solution is between 40 and 60 g/Lbefore further concentration by ultrafiltration.

93. A method of decreasing the difference between a calculated orestimated antibody concentration and an actual measured antibodyconcentration in a highly concentrated antibody ultrafiltrationretentate to less than 20%, wherein sucrose is added to the solutionwhen the antibody concentration of the solution is between 40 and 60 g/Lbefore further concentration by ultrafiltration.

94. A method of decreasing the difference between a calculated antibodyor estimated concentration and an actual measured antibody concentrationin a highly concentrated antibody ultrafiltration retentate to less than25%, wherein sucrose is added to the solution when the antibodyconcentration of the solution is between 40 and 60 g/L before furtherconcentration by ultrafiltration.

95. A method of decreasing the difference between a calculated orestimated antibody concentration and an actual measured antibodyconcentration in a highly concentrated antibody ultrafiltration to lessthan 30%, wherein sucrose is added to the solution when the antibodyconcentration of the solution is between 40 and 60 g/L before furtherconcentration by ultrafiltration.

96. A method of ultrafiltration of a highly concentrated antibodysolution, where the difference between the calculated or estimatedantibody concentration and the actual measured antibody concentration inthe highly concentrated ultrafiltration retentate is less than 15%,wherein sucrose is added to the solution when the antibody concentrationof the solution is between 40 and 60 g/L before further concentration byultrafiltration.

97. A method of ultrafiltration of a highly concentrated antibodysolution, where the difference between the calculated or estimatedantibody concentration and the actual measured antibody concentration inthe highly concentrated ultrafiltration retentate is less than 20%,wherein sucrose is added to the solution when the antibody concentrationof the solution is between 40 and 60 g/L before further concentration byultrafiltration.

98. A method of ultrafiltration of a highly concentrated antibodysolution, where the difference between the calculated or estimatedantibody concentration and the actual measured antibody concentration inthe highly concentrated ultrafiltration retentate is less than 25%,wherein sucrose is added to the solution when the antibody concentrationof the solution is between 40 and 60 g/L before further concentration byultrafiltration.

99. A method of ultrafiltration of a highly concentrated antibodysolution, where the difference between the calculated or estimatedantibody concentration and the actual measured antibody concentration inthe highly concentrated ultrafiltration retentate is less than 30%,wherein sucrose is added to the solution when the antibody concentrationof the solution is between 40 and 60 g/L before further concentration byultrafiltration.

100. A method of increasing the recovery of antibody by ultrafiltrationof a highly concentrated antibody solution, wherein sucrose is added tothe solution when the antibody concentration of the solution is between40 and 60 g/L before further concentration by ultrafiltration.

101. A method of increasing the recovery of antibody by ultrafiltrationof a highly concentrated antibody solution to above 94% of the totalamount of said antibody in the solution to be ultrafiltrated, whereinsucrose is added to the solution when the antibody concentration of thesolution is between 40 and 60 g/L before further concentration byultrafiltration.

102. A method of increasing the recovery of antibody by ultrafiltrationof a highly concentrated antibody solution to above 95% of the totalamount of said antibody in the solution to be ultrafiltrated, whereinsucrose is added to the solution when the antibody concentration of thesolution is between 40 and 60 g/L before further concentration byultrafiltration.

103. A method of increasing the recovery of antibody by ultrafiltrationof a highly concentrated antibody solution to above 96% of the totalamount of said antibody in the solution to be ultrafiltrated, whereinsucrose is added to the solution when the antibody concentration of thesolution is between 40 and 60 g/L before further concentration byultrafiltration.

104. A method of increasing the recovery of antibody by ultrafiltrationof a highly concentrated antibody solution to above 97% of the totalamount of said antibody in the solution to be ultrafiltrated, whereinsucrose is added to the solution when the antibody concentration of thesolution is between 40 and 60 g/L before further concentration byultrafiltration.

105. A method of increasing the recovery of antibody by ultrafiltrationof a highly concentrated antibody solution to above 98% of the totalamount of said antibody in the solution to be ultrafiltrated, whereinsucrose is added to the solution when the antibody concentration of thesolution is between 40 and 60 g/L before further concentration byultrafiltration.

106. A method of increasing the recovery of antibody by ultrafiltrationof a highly concentrated antibody solution to above 99% of the totalamount of said antibody in the solution to be ultrafiltrated, whereinsucrose is added to the solution when the antibody concentration of thesolution is between 40 and 60 g/L before further concentration byultrafiltration.

107. A method of ultrafiltration of a highly concentrated antibodysolution, where the recovery of antibody by ultrafiltration is above 95%of the total amount of said antibody in the solution to beultrafiltrated, wherein sucrose is added to the solution when theantibody concentration of the solution is between 40 and 60 g/L beforefurther concentration by ultrafiltration.

107. A method of ultrafiltration of a highly concentrated antibodysolution, where the recovery of antibody by ultrafiltration is above 96%of the total amount of said antibody in the solution to beultrafiltrated, wherein sucrose is added to the solution when theantibody concentration of the solution is between 40 and 60 g/L beforefurther concentration by ultrafiltration.

109. A method of ultrafiltration of a highly concentrated antibodysolution, where the recovery of antibody by ultrafiltration is above 97%of the total amount of said antibody in the solution to beultrafiltrated, wherein sucrose is added to the solution when theantibody concentration of the solution is between 40 and 60 g/L beforefurther concentration by ultrafiltration.

110. A method of ultrafiltration of a highly concentrated antibodysolution, where the recovery of antibody by ultrafiltration is above 98%of the total amount of said antibody in the solution to beultrafiltrated, wherein sucrose is added to the solution when theantibody concentration of the solution is between 40 and 60 g/L beforefurther concentration by ultrafiltration.

111. A method of ultrafiltration of a highly concentrated antibodysolution, where the recovery of antibody by ultrafiltration is above 99%of the total amount of said antibody in the solution to beultrafiltrated, wherein sucrose is added to the solution when theantibody concentration of the solution is between 40 and 60 g/L beforefurther concentration by ultrafiltration.

112. A method of increasing the recovery of antibody by ultrafiltrationof a highly concentrated antibody solution in the final ultrafiltrationretentate to above 50% of the total amount of said antibody in thesolution to be ultrafiltrated, wherein sucrose is added to the solutionwhen the antibody concentration of the solution is between 40 and 60 g/Lbefore further concentration by ultrafiltration.

113. A method of increasing the recovery of antibody by ultrafiltrationof a highly concentrated antibody solution in the final ultrafiltrationretentate to above 55% of the total amount of said antibody in thesolution to be ultrafiltrated, wherein sucrose is added to the solutionwhen the antibody concentration of the solution is between 40 and 60 g/Lbefore further concentration by ultrafiltration.

114. A method of increasing the recovery of antibody by ultrafiltrationof a highly concentrated antibody solution in the final ultrafiltrationretentate to above 60% of the total amount of said antibody in thesolution to be ultrafiltrated, wherein sucrose is added to the solutionwhen the antibody concentration of the solution is between 40 and 60 g/Lbefore further concentration by ultrafiltration.

115. A method of ultrafiltration of a highly concentrated antibodysolution, where the recovery of antibody by ultrafiltration in the finalultrafiltration retentate is above 50% of the total amount of saidantibody in the solution to be ultrafiltrated, wherein sucrose is addedto the solution when the antibody concentration of the solution isbetween 40 and 60 g/L before further concentration by ultrafiltration.

116. A method of ultrafiltration of a highly concentrated antibodysolution, where the recovery of antibody by ultrafiltration in the finalultrafiltration retentate is above 55% of the total amount of saidantibody in the solution to be ultrafiltrated, wherein sucrose is addedto the solution when the antibody concentration of the solution isbetween 40 and 60 g/L before further concentration by ultrafiltration.

117. A method of ultrafiltration of a highly concentrated antibodysolution, where the recovery of antibody by ultrafiltration in the finalultrafiltration retentate is above 60% of the total amount of saidantibody in the solution to be ultrafiltrated, wherein sucrose is addedto the solution when the antibody concentration of the solution isbetween 40 and 60 g/L before further concentration by ultrafiltration.

118. A method of stabilizing a highly concentrated antibody solutionduring ultrafiltration, wherein sucrose is added to the solution whenthe antibody concentration of the solution is between 40 and 60 g/Lbefore further concentration by ultrafiltration.

119. A method of stabilizing a highly concentrated antibody solutionduring ultrafiltration, where the level of HMWP aggregates is 1% orbelow 1%, wherein sucrose is added to the solution when the antibodyconcentration of the solution is between 40 and 60 g/L before furtherconcentration by ultrafiltration.

120. A method of stabilizing a highly concentrated antibody solutionduring ultrafiltration, where the level of HMWP aggregates is below1.1%, wherein sucrose is added to the solution when the antibodyconcentration of the solution is between 40 and 60 g/L before furtherconcentration by ultrafiltration.

121. A method of decreasing the formation of HWMP aggregates in a highlyconcentrated antibody solution during ultrafiltration, wherein sucroseis added to the solution when the antibody concentration of the solutionis between 40 and 60 g/L before further concentration byultrafiltration.

122. A method of decreasing the formation of HWMP aggregates in a highlyconcentrated antibody solution during ultrafiltration to 1% or below 1%,wherein sucrose is added to the solution when the antibody concentrationof the solution is between 40 and 60 g/L before further concentration byultrafiltration.

123. A method of decreasing the formation of HWMP aggregates in a highlyconcentrated antibody solution during ultrafiltration to below 1.1%,wherein sucrose is added to the solution when the antibody concentrationof the solution is between 40 and 60 g/L before further concentration byultrafiltration.

124. A method of ultrafiltration of a highly concentrated antibodysolution, where the level of HMWP aggregates is kept at 1% or below 1%,wherein sucrose is added to the solution when the antibody concentrationof the solution is between 40 and 60 g/L before further concentration byultrafiltration.

125. A method of ultrafiltration of a highly concentrated antibodysolution, where the level of HMWP aggregates is kept below 1.1%, whereinsucrose is added to the solution when the antibody concentration of thesolution is between 40 and 60 g/L before further concentration byultrafiltration.

126. A method of stabilizing a highly concentrated antibody solutionduring ultrafiltration, where the formation of HMWP aggregates duringultrafiltration is decreased by 10%, wherein sucrose is added to thesolution when the antibody concentration of the solution is between 40and 60 g/L before further concentration by ultrafiltration.

127. A method of stabilizing a highly concentrated antibody solutionduring ultrafiltration, where the formation of HMWP aggregates duringultrafiltration is decreased by 20%, wherein sucrose is added to thesolution when the antibody concentration of the solution is between 40and 60 g/L before further concentration by ultrafiltration.

128. A method of ultrafiltration of a highly concentrated antibodysolution, where the formation of HMWP aggregates is decreased by 10%,wherein sucrose is added to the solution when the antibody concentrationof the solution is between 40 and 60 g/L before further concentration byultrafiltration.

129. A method of ultrafiltration of a highly concentrated antibodysolution, where the formation of HMWP aggregates is decreased by 20%,wherein sucrose is added to the solution when the antibody concentrationof the solution is between 40 and 60 g/L before further concentration byultrafiltration.

130. A method according to any of embodiments 90-129, wherein theantibody concentration is 40 g/L when sucrose is added to the solution.

131. A method according to any of embodiments 90-129, wherein theantibody concentration is 41 g/L when sucrose is added to the solution.

132. A method according to any of embodiments 90-129, wherein theantibody concentration is 42 g/L when sucrose is added to the solution.

133. A method according to any of embodiments 90-129, wherein theantibody concentration is 43 g/L when sucrose is added to the solution.

134. A method according to any of embodiments 90-129, wherein theantibody concentration is 44 g/L when sucrose is added to the solution.

135. A method according to any of embodiments 90-129, wherein theantibody concentration is 45 g/L when sucrose is added to the solution.

136. A method according to any of embodiments 90-129, wherein theantibody concentration is 46 g/L when sucrose is added to the solution.

137. A method according to any of embodiments 90-129, wherein theantibody concentration is 47 g/L when sucrose is added to the solution.

138. A method according to any of embodiments 90-129, wherein theantibody concentration is 48 g/L when sucrose is added to the solution.

139. A method according to any of embodiments 90-129, wherein theantibody concentration is 49 g/L when sucrose is added to the solution.

140. A method according to any of embodiments 90-129, wherein theantibody concentration is 50 g/L when sucrose is added to the solution.

141. A method according to any of embodiments 90-129, wherein theantibody concentration is 51 g/L when sucrose is added to the solution.

142. A method according to any of embodiments 90-129, wherein theantibody concentration is 52 g/L when sucrose is added to the solution.

143. A method according to any of embodiments 90-129, wherein theantibody concentration is 53 g/L when sucrose is added to the solution.

144. A method according to any of embodiments 90-129, wherein theantibody concentration is 54 g/L when sucrose is added to the solution.

145. A method according to any of embodiments 90-129, wherein theantibody concentration is 55 g/L when sucrose is added to the solution.

146. A method according to any of embodiments 90-129, wherein theantibody concentration is 56 g/L when sucrose is added to the solution.

147. A method according to any of embodiments 90-129, wherein theantibody concentration is 57 g/L when sucrose is added to the solution.

148. A method according to any of embodiments 90-129, wherein theantibody concentration is 58 g/L when sucrose is added to the solution.

149. A method according to any of embodiments 90-129, wherein theantibody concentration is 59 g/L when sucrose is added to the solution.

150. A method according to any of embodiments 90-129, wherein theantibody concentration is 60 g/L when sucrose is added to the solution.

151. A method according to any of embodiments 90-129, wherein theantibody concentration is between 40 g/L and 45 g/L when sucrose isadded to the solution.

152. A method according to any of embodiments 90-129, wherein theantibody concentration is between 40 g/L and 50 g/L when sucrose isadded to the solution.

153. A method according to any of embodiments 90-129, wherein theantibody concentration is between 45 g/L and 50 g/L when sucrose isadded to the solution.

154. A method according to any of embodiments 90-129, wherein theantibody concentration is between 45 g/L and 55 g/L when sucrose isadded to the solution.

155. A method according to any of embodiments 90-129, wherein theantibody concentration is between 50 g/L and 55 g/L when sucrose isadded to the solution.

156. A method according to any of embodiments 90-129, wherein theantibody concentration is between 50 g/L and 60 g/L when sucrose isadded to the solution.

157. A method according to any of embodiments 90-129, wherein theantibody concentration is between 55 g/L and 60 g/L when sucrose isadded to the solution.

158. A method according to any of embodiments 90-157, wherein thesucrose is added in the concentration of between 50 mM and 300 mM.

159. A method according to any of embodiments 90-157, wherein thesucrose is added in the concentration of between 50 mM and 250 mM.

160. A method according to any of embodiments 90-157, wherein thesucrose is added in the concentration of between 50 mM and 200 mM.

161. A method according to any of embodiments 90-157, wherein thesucrose is added in the concentration of between 50 mM and 150 mM.

162. A method according to any of embodiments 90-157, wherein thesucrose is added in the concentration of between 50 mM and 100 mM.

163. A method according to any of embodiments 90-157, wherein thesucrose is added in the concentration of between 100 mM and 300 mM.

164. A method according to any of embodiments 90-157, wherein thesucrose is added in the concentration of between 100 mM and 250 mM.

165. A method according to any of embodiments 90-157, wherein thesucrose is added in the concentration of between 100 mM and 200 mM.

166. A method according to any of embodiments 90-157, wherein thesucrose is added in the concentration of between 100 mM and 150 mM.

167. A method according to any of embodiments 90-157, wherein thesucrose is added in the concentration of 100 mM.

168. A method according to any of embodiments 90-157, wherein thesucrose is added in the concentration of 110 mM.

169. A method according to any of embodiments 90-157, wherein thesucrose is added in the concentration of 120 mM.

170. A method according to any of embodiments 90-157, wherein thesucrose is added in the concentration of 130 mM.

171. A method according to any of embodiments 90-157, wherein thesucrose is added in the concentration of 140 mM.

172. A method according to any of embodiments 90-157, wherein thesucrose is added in the concentration of 150 mM.

173. A method according to any of embodiments 90-157, wherein thesucrose is added in the concentration of 160 mM.

174. A method according to any of embodiments 90-157, wherein thesucrose is added in the concentration of 175 mM.

175. A method according to any of embodiments 90-157, wherein thesucrose is added in the concentration of 200 mM.

176. A method according to any of embodiments 90-157, wherein thesucrose is added in the concentration of 225 mM.

177. A method according to any of embodiments 90-157, wherein thesucrose is added in the concentration of 250 mM.

178. A method according to any of embodiments 90-157, wherein thesucrose is added in the concentration of 300 mM.

179. A method according to any of embodiments 90-178, wherein theantibody is a monoclonal antibody.

180. A method according to any of embodiments 90-179, wherein theantibody is of the IgG4 subtype.

181. A method according to any of embodiments 90-180, wherein theantibody is a human antibody.

182. A method according to any of embodiments 90-180, wherein theantibody is a humanized antibody.

183. A method according to any of embodiments 90-182, wherein theantibody is an anti-IL-20 monoclonal antibody.

184. A method of concentrating a protein in a protein solution accordingto any of embodiments 1-89, for use in a pharmaceutical composition.

185. A method of concentrating a protein in a protein solution accordingto any of embodiments 1-89, for use for manufacture of a medicament.

186. A method of treating an inflammatory disease which comprisesadministering to a patient the pharmaceutical composition of embodiment184.

187. A method of treating an inflammatory disease which comprisesadministering to a patient the medicament of embodiment 185.

188. A method of concentrating an antibody in an antibody solutionaccording to any of embodiments 90-183, for use in a pharmaceuticalcomposition.

189. A method of concentrating an antibody in an antibody solutionaccording to any of embodiments 90-183, for use for manufacture of amedicament.

190. A method of treating an inflammatory disease which comprisesadministering to a patient the pharmaceutical composition of embodiment188.

191. A method of treating an inflammatory disease which comprisesadministering to a patient the medicament of embodiment 189.

192. A method of concentrating a highly concentrated protein solutionwhich comprises:

(a) adding sucrose to the highly concentrated protein solution when theprotein concentration is between 40 g/L and 60 g/L;

(b) concentrate the highly concentrated protein solution byultrafiltration.

193. A method of decreasing the difference between a calculated andmeasured protein concentration in a highly concentrated proteinultrafiltration retentate which comprises:

(a) adding sucrose to the highly concentrated protein solution when theprotein concentration is between 40 g/L and 60 g/L;

(b) concentrate the highly concentrated protein solution byultrafiltration.

194: A method of decreasing the difference between a calculated andmeasured protein concentration in a highly concentrated proteinultrafiltration retentate which comprises:

(a) adding sucrose to a highly concentrated protein solution when theprotein concentration is between 40 g/L and 60 g/L

(b) concentrate the highly concentrated protein solution byultrafiltration.

195: A method of decreasing the difference between a calculated andmeasured protein concentration to less than 30% in a highly concentratedprotein ultrafiltration retentate which comprises:

(a) adding sucrose to a highly concentrated protein solution when theprotein concentration is between 40 g/L and 60 g/L

(b) concentrate the highly concentrated protein solution byultrafiltration.

196. A method of increasing the recovery of protein by ultrafiltrationof a highly concentrated protein solution which comprises:

(a) adding sucrose to the highly concentrated protein solution when theprotein concentration is between 40 g/L and 60 g/L

(b) concentrate the highly concentrated protein solution byultrafiltration.

197. A method of increasing the recovery of protein by ultrafiltrationof a highly concentrated protein solution to above 50% of the totalamount of said protein in the highly concentrated solution to beultrafiltrated which comprises:

(a) adding sucrose to the highly concentrated protein solution when theprotein concentration is between 40 g/L and 60 g/L

(b) concentrate the highly concentrated protein solution byultrafiltration.

198. A method of stabilizing a highly concentrated protein solutionduring ultrafiltration which comprises:

(a) adding sucrose to the highly concentrated protein solution when theprotein concentration is between 40 g/L and 60 g/L

(b) concentrate the highly concentrated protein solution byultrafiltration.

199. A method of suppressing the formation of HMWP duringultrafiltration of a highly concentrated protein solution whichcomprises:

(a) adding sucrose to the highly concentrated protein solution when theprotein concentration is between 40 g/L and 60 g/L

(b) concentrate the highly concentrated protein solution byultrafiltration.

200. A method of ultrafiltration of a highly concentrated proteinsolution where the level of HMWP is suppressed to 1% or below 1% whichcomprises:

(a) adding sucrose to the highly concentrated protein solution when theprotein concentration is between 40 g/L and 60 g/L

(b) concentrate the highly concentrated protein solution byultrafiltration.

201. A method of concentrating a highly concentrated antibody solutionwhich comprises:

(a) adding sucrose to the highly concentrated antibody solution when theantibody concentration is between 40 g/L and 60 g/L;

(b) concentrate the highly concentrated antibody solution byultrafiltration.

202. A method of decreasing the difference between a calculated andmeasured antibody concentration in a highly concentrated antibodyultrafiltration retentate which comprises:

(a) adding sucrose to the highly concentrated antibody solution when theantibody concentration is between 40 g/L and 60 g/L;

(b) concentrate the highly concentrated antibody solution byultrafiltration.

203: A method of decreasing the difference between a calculated andmeasured antibody concentration in a highly concentrated antibodyultrafiltration retentate which comprises:

(a) adding sucrose to a highly concentrated antibody solution when theantibody concentration is between 40 g/L and 60 g/L

(b) concentrate the highly concentrated antibody solution byultrafiltration.

204: A method of decreasing the difference between a calculated andmeasured antibody concentration to less than 30% in a highlyconcentrated antibody ultrafiltration retentate which comprises:

(a) adding sucrose to a highly concentrated antibody solution when theantibody concentration is between 40 g/L and 60 g/L

(b) concentrate the highly concentrated antibody solution byultrafiltration.

205. A method of increasing the recovery of antibody by ultrafiltrationof a highly concentrated antibody solution which comprises:

(a) adding sucrose to the highly concentrated antibody solution when theantibody concentration is between 40 g/L and 60 g/L

(b) concentrate the highly concentrated antibody solution byultrafiltration.

206. A method of increasing the recovery of antibody by ultrafiltrationof a highly concentrated antibody solution to above 50% of the totalamount of said antibody in the highly concentrated solution to beultrafiltrated which comprises:

(a) adding sucrose to the highly concentrated antibody solution when theantibody concentration is between 40 g/L and 60 g/L

(b) concentrate the highly concentrated antibody solution byultrafiltration.

207. A method of stabilizing a highly concentrated antibody solutionduring ultrafiltration which comprises:

(a) adding sucrose to the highly concentrated antibody solution when theantibody concentration is between 40 g/L and 60 g/L

(b) concentrate the highly concentrated antibody solution byultrafiltration.

208. A method of suppressing the formation of HMWP duringultrafiltration of a highly concentrated antibody solution whichcomprises:

(a) adding sucrose to the highly concentrated antibody solution when theantibody concentration is between 40 g/L and 60 g/L

(b) concentrate the highly concentrated antibody solution byultrafiltration.

209. A method of ultrafiltration where the level of HMWP is suppressedto 1% or below 1% which comprises:

(a) adding sucrose to the highly concentrated antibody solution when theantibody concentration is between 40 g/L and 60 g/L

(b) concentrate the highly concentrated antibody solution byultrafiltration.

210. A method according to any of embodiments 192-209, wherein theprotein or antibody concentration is between 45 g/L and 55 g/L whensucrose is added to the solution.

211. A method according to any of embodiments 192-210, wherein thesucrose is added in the concentration of between 100 mM and 300 mM.

While certain features of the invention have been illustrated anddescribed herein, many modifications, substitutions, changes, andequivalents will now occur to those of ordinary skill in the art. It is,therefore, to be understood that the appended claims are intended tocover all such modifications and changes as fall within the true spiritof the invention.

1. A method of ultrafiltration of a highly concentrated proteinsolution, wherein sucrose is added to the solution when the proteinconcentration of the solution is between 40 and 60 g/L before furtherconcentration by ultrafiltration.
 2. The method according to claim 1,wherein the difference between the calculated protein concentration andthe measured protein concentration in the highly concentratedultrafiltration retentate is decreased.
 3. The method according to claim1, wherein the difference between the calculated protein concentrationand the measured protein concentration in the highly concentratedultrafiltration retentate is less than 30%.
 4. The method according toclaim 1, wherein the addition of sucrose stabilizes the highlyconcentrated solution to be ultrafiltrated.
 5. The method according toclaim 1, wherein the level of HMWP aggregates is 1% or below 1% in thehighly concentrated ultrafiltration retentate.
 6. The method accordingto claim 1, wherein the recovery of the protein during ultrafiltrationis increased.
 7. The method according to claim 1, wherein the recoveryof the protein during ultrafiltration is 94% or above, of the totalamount of protein in the highly concentrated protein solution to beultrafiltrated.
 8. The method according to claim 1, wherein the sucroseis added to the highly concentrated protein solution in theconcentration of between 50 mM and 300 mM.
 9. The method according toclaim 1, wherein the protein is an antibody.
 10. The method according toclaim 1, wherein the antibody is a monoclonal antibody.
 11. The methodaccording to claim 10, wherein the antibody is an anti-IL-20 monoclonalantibody.
 12. The method according to claim 2, wherein the differencebetween the calculated protein concentration and the measured proteinconcentration in the highly concentrated ultrafiltration retentate isless than 30%.
 13. A pharmaceutical composition comprising a protein oran antibody concentrated from a protein or antibody solution by usingthe method of claim
 1. 14. A method of treating an inflammatory diseasewhich comprises administering to a patient the pharmaceuticalcomposition of claim 13.